In the fields of medicine and clinical chemistry, many studies and determinations of physiologically reactive species such as cells, proteins, enzymes, cofactors, nucleic acids, substrates, antigens, antibodies, and so forth are carried out using conjugates involving specific binding pair members or labels or the like. Various assay techniques that involve the binding of specific binding pair members are known. These assay techniques generally also involve a label used in the detection part of the assay.
The use of enzyme labels for the determination of analytes in immunoassays has shown substantial promise and numerous immunoassays have been developed, which are dependent upon the accurate measurement of enzymatic activity from an assay medium.
Numerous enzyme immunoassay methods are known for the determination of the presence in a sample of an analyte. The assays include both homogeneous and heterogeneous assays. Some homogeneous approaches comprise determining the effect that the sample containing the analyte has on the binding between a conjugate of the analyte (ligand) and an enzyme and a receptor for the analyte. When the conjugate and the receptor bind, a modulation of the enzymatic activity occurs. The presence of analyte in the sample can be determined from the effect that the analyte has on the modulation of the enzymatic activity when compared to that obtained in the absence of analyte or in the presence of known amounts of analyte. Heterogeneous assays include sandwich format assays. The aforementioned assays are discussed in more detail below.
Polypeptides can be conjugated with enzymes using preactivated enzymes such as, for example, enzymes preactivated with a maleimide or a haloacetyl moiety. The polypeptides may comprise free sulfhydryl (—SH) groups or may be treated to introduce such groups. In the latter circumstance, linking groups may be employed to link to the polypeptide to introduce sulfhydryl groups. Accordingly, the linking group may have an amine-reactive functionality in addition to the sulfhydryl group where the amine reactive functionality reacts with free amine groups on the polypeptide. Maleimide-activated enzyme is reacted with free sulfhydryl (—SH) groups present in, or introduced into, the polypeptide to form a stable thiol ether linkage.
In another approach, amino groups of a polypeptide are acylated with a heterobifunctional crosslinking agent such as N-succinimidyl 4′-(p-maleimidophenyl) butyrate. This crosslinking agent has a N-hydroxysuccinimide group at one end of the molecule that reacts with amino groups. The maleimide moiety at the other end of the heterobifunctional agent reacts with free sulfhydryl groups of a polypeptide.
In the above reactions, it is standard practice to terminate the conjugation reaction with a quench reagent that deactivates one of the reactive groups that is used to form the conjugate, namely, free sulfhydryl groups remaining on the product after the conjugation reaction has taken place. The sulfhydryl groups are deactivated by adding a reagent reactive with the sulfhydryl groups such as, for example, a sulfhydryl receptor, e.g., a maleimide, and so forth.
There remains a need for methods for producing ligand binder-polypeptide conjugates such as, for example, enzyme-polypeptide conjugates, polynucleotide-polypeptide conjugates, and so forth, that exhibit good stability, activity, sensitivity, and the like.